Gene expression is regulated by genetic and epigenetic mechanisms. There are a variety of epigenetic mechanisms including DNA methylation (at CpG dinucleotides) and nucleosome positioning, which work together to generate chromatin states. Specific chromatin states facilitate, inhibit or allow for the potential of gene activation. Genome wide studies of chromatin states have focused on either DNA methylation or nucleosome positioning, and as a result a comprehensive integrated genome-wide view of DNA methylation and nucleosome positioning has not been done.
Methylation dependent single molecule footprinting techniques (M-SPA) rely on CpG methylation. Since CpG methylation occurs endogenously, analysis is limited to regions that are unmethylated. In addition, CpG sites are predisposed to mutation and thus have become under-represented in the genome overall and asymmetrically distributed into CpG rich and CpG poor regions. Thus M-SPA is limited to regions that are CpG rich. GpC dinucleotides do not have the same propensity for mutation and are more broadly distributed throughout in the genome.
As such, there is a continuing need for improved methods for determining endogenous methylation and nucleosome positioning simultaneously.
Recently, a GpC methyltransferase enzyme M.CviPI has become commercially available. M.CviPI methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence 5′ . . . GC . . . 3′. M.CviPI is isolated from a strain of E. coli which contains the methyltransferase gene from Chlorella virus. This construct is fused to the maltose binding protein (MBP).